The Cytotoxic Cell Studies Group has made significant advances in understanding the molecular mechanism of natural killer (NK) cell function by examining the role of a novel gene, Nktr, in the tumor recognition/killing mechanism of NK cells. The crucial role of the NK-TR protein in target cell recognition/killing was demonstrated by inhibiting NK-TR expression in primary human and mouse NK cells with a retroviral vector producing antisense Nktr RNA. Experiments with freshly isolated human and mouse NK cells have demonstrated a dramatic loss of NK activity relative to control retrovirus infections. Although NK cells are the only freshly isolated cell to express NK-TR, several non-NK cell lines were shown to be positive. A burst of NK-TR expression was observed when the human HL-60 cell line was induced to differentiate into granulocytes. This finding lead to the study of the role of the NK-TR in the process of hematopoietic differentiation. Antisense Nktr transfectants of the HL-60 cell line were no longer able to differentiate into granulocytes. The possible involvement of NK-TR in pre-mRNA splicing has been strengthened by our discovery of an NK-TR related protein (CARS-Cyp) that is expressed in every tissue and cell type examined with the exception of NK cells. The rat homolog of CARS-Cyp has been identified as a nuclear protein associated with splicing complexes. Our current studies are directed towards defining NK-TR function in cytotoxic cells and developing gene knockout mice. Mice heterozygous for an Nktr null mutation have been generated, and are currently being bred to produce homozygous Nktr -deficient mice. The Nktr promoter has been analyzed, and it does not repre-sent an NK cell-specific promotor. The NK specificity of NK-TR expression was shown to be controlled at the level of pre-mRNA splicing. We have recently cloned two members of the Ly-49 family that are expressed on murine NK cells. Ly-49G2, which appears to recognize the class I molecules H2-Dd and/ or H2-Ld and Ly-49D whose class I recognition is under study. The previously studied Ly-49 family members are inhibitory receptors that recognize MHC class I, however, Ly-49D has been shown to represent